Detection and isolation of cell populations from muscle using antibodies to fa1/dlk1

ABSTRACT

The present invention relates to the use of antibodies recognizing Fetal Antigen-1 (FA1/dlk1) for the detection and isolation of cell populations in mammalian muscle. In one embodiment, myogenic progenitor cells are detected in developing, diseased or regenerating muscle. In another embodiment, muscle stem and progenitor myogenic progenitor cells are isolated from muscle tissue or from cultures containing muscle cells. The isolated cells may be used for transplantation, drug screening, production of cell type specific antibodies, and gene therapy and discovery. Transplantation of these cells may provide treatments for degenerative diseases of muscle, and for regeneration of muscle following trauma or ischemia such as myocardial infarction.

The present invention is a non-provisional of U.S.-provisional patentapplication Ser. No. 60/366,421 filed on 21 Mar. 2002 and claimspriority from Danish patent application no. PA 2002 00481 filed on 27Mar. 2002. All references cited in these applications or in the presentapplication are hereby incorporated by reference in their entirety.

FIELD OF INVENTION

The present invention concerns the use of FA1 antibodies for recognizingand isolating FA-1 expressing cells derived from mammalian muscle, whichincludes myogenic stem/progenitor cells.

BACKGROUND OF THE INVENTION

Skeletal muscles of adult mammalian species exhibit a capacity to adaptto physiological demands such as growth, training, and injury. Theprocesses by which these adaptations occur are attributed to a smallpopulation of mononuclear cells that is resident in adult skeletalmuscle and has been referred to as satellite cells. Myogenic satellitecells have been the subject of a recent extensive review (Hawke & Garry2001. J Appl Physiol 91: 534-551) Skeletal muscle fibers are terminallydifferentiated and the nuclei in these multinucleated cells areincapable of DNA synthesis or mitotic division. Increases in musclefiber numbers or in numbers of muscle fiber nuclei are due toproliferation and subsequent differentiation of muscle precursor cellsknown as “myoblasts.” In adults, myoblasts remain as mitoticallyquiescent reserve precursor populations, which can, upon muscle injury,re-enter the cell cycle, undergo several rounds of proliferation, andsubsequently differentiate and permanently exit from the cell cycle.Upon differentiation, differentiated myoblasts acquire the ability tofuse with one another or with preexisting muscle fibers, and alsocommence expression of a set of muscle-specific myofibrillary andcontractile proteins. Quiescent myogenic progenitor cells are physicallydistinct from the adult myofibers as they reside in indentations betweenthe sarcolemma and the basal lamina. In the case of muscle injury, someof these cells will remain as progenitor cells whereas others willdifferentiate into new muscle fibers. In response to stimuli such asmyotrauma, myogenic progenitor cells become activated, proliferate, andexpress myogenic markers. Ultimately, these cells fuse to existingmuscle fibers or fuse together to form new myofibers during regenerationof damaged skeletal muscle. Gussoni, (Nature 1999 Sep.23;401(6751):390-39) found that intravenous injection of a population ofmuscle-derived stem cells into a mouse model of Duchenne's musculardystrophy results in the incorporation of donor-derived nuclei intomuscle, and the partial restoration of dystrophin expression in theaffected muscle. The population containing the muscle stem cells wasisolated by its low affinity for a particular dye. This intravenousroute of administration may represent a means of treating degenerativediseases of muscle with a suitable population of myogenic progenitorcells.

Cardiac muscle, unlike skeletal muscle does not have the capacity toregenerate in response to injury. Populations of myogenic progenitorcells could therefore be used to regenerate cardiac muscle followingmyocardial infarction or ischemia (El Oakley R M Ann Thorac Surg 2001May; 71(5): 1724-1733). The plasticity of the myogenic progenitor cellpopulation suggests that these cells not only have a capacity for muscleregeneration but may also contribute to non-muscle lineages (Springer ML, et al. J Clin Invest. 2001 June; 107(11): 1355-6).

Some potential markers have been associated with myogenic progenitorcells. Satellite cells have been shown to express neural cell adhesionmolecule (N-CAM) (Fidzianska A Folia Neuropathol 1995; 33(3): 125-128).Magnetic affinity cell sorting (MACS) has been used in the separation ofNCAM-positive, cultured myogenic cells from normal and dystrophic dogs(Prattis S M Exp Cell Res 1993 October; 208(2): 453-64). Vascularadhesion molecule-1 (VCAM-1) is expressed on satellite cells andregenerating muscle fibres. (Jesse T L. J Cell Biol 140: 1265-1276,1998) and M-cadherin, calcium-dependent cell adhesion molecule, has beensuggested as a marker of the satellite cell population (Irintchev. DevDyn 199: 326-337, 1994). Similarly, Bcl-2 has been shown to be expressedin muscle stem cells (U.S. Pat. No. 6,337,184). Pax7 is a further makerof myogenic progenitor cells whose location is restricted to asub-population of satellite cells (Seale P et al. Cell2000;102:777-786).

The present invention provides a means of identifying and enriching forsubpopulations of mononuclear myogenic progenitor cells that expressFA1/dlk1 with or without selection for other markers.

Fetal antigen 1 (FA1) is one of the increasing numbers of proteinsbelonging to the epidermal growth factor (EGF)-super family that havebeen identified within the last decade. The protein contains 6 EGF-likerepeats and displays a very similar primary structure and level ofglycosylation in man, mouse and rat (Jensen C H, et al. Hum Reprod 19938(4), 635-641; Jensen C H, et al. Eur J Biochem 1994 225(1), 83-92.Bachmann E, et al. J Reprod Fertil 1996 107(2), 279-285. Krogh T N, etal. Eur J Biochem 1997 244(2), 334-342. Carlsson H E, et al. Biol Reprod2000 63(1), 30-33.)

FA1 is synthesized as a larger transmembrane precursor and released fromcells after proteolytic action of an unidentified enzyme. Several groupshave described cDNA clones for this precursor, each assigning a new namefor the cDNA depending on the species and tissue/cell type from whichthey isolated it. As a result, the FA1 precursor has been referred to asadrenal specific mRNA (human pG2 Helman L J. Nucleic Acids Res 199018(3), 685), delta-like (mouse and human dlk1 Laborda J, et al. J BiolChem 1993 268(6), 3817-3820.), preadipocyte factor-1 (mouse, rat andbovine pref-1 Laborda J, et al. J Biol Chem 1993 268(6), 3817-3820. SmasC M, Cell 1993 73(4), 725-734; Carlsson C, et al. Endocrinology 1997138(9), 3940; Fahrenkrug S C, Biochem Biophys Res Commun 1999 264(3),662-667) and zona glomerulosa-specific factor (rat ZOG Okamoto, et al.Steroids 1997 62(1), 73-76). The official name for the gene encodingthis membrane-associated protein is now delta-homologue 1, dlk11(Gubina, et al. Cytogenet Cell Genet 1999 84(3-4), 206-207.), referringto the close resemblance between its EGF-repeats and those of thetransmembrane protein Delta, which was originally described inDrosophila Melanogaster. Delta is one of the ligands for the Notchreceptor and interactions between these membrane proteins are crucialfor the development of various tissues [Artavanis-Tsakonas, Science 1995268(5208), 225-232.]. The primary structure of dlk1 does not allowconclusions as to whether it is a ligand or receptor, but both themembrane-associated and the soluble form (i.e. FA1) of the DLK1 genehave been shown to be involved in the differentiation/proliferationprocesses of various cell types and act through autocrine/paracrine andjuxtacrine intercellular signaling (reviewed by Laborda in Laborda J.Histol Histopathol 2000 15(1), 119-129.), the membrane-associated formpossibly as a homodimer (Kaneta, J Immunol 2000 164(1), 256-264). Apartfrom being present in preadipocytes and stromal cells, the expression ofFA1/dlk1 in adults seems to be associated with endocrine structures. FA1has been localized in β-cells of the pancreatic islets of Langerhans(Jensen, Hum Reprod 1993 8(4), 635-641); Jensen, Eur J Biochem 1994225(1), 83-92; Tornehave, Histochem Cell Biol 1996 106(6), 535], theadrenal gland (medulla and cortex) [Jensen, Hum Reprod 1993 8(4),635-641], the somatotroph cells of the adenopituitary gland [Larsen,Lancet 1996 347(8995), 191], the sex hormone-producing Leydig cells ofthe testis, and theca interna and Hilus cells of the ovary [Jensen, MolHum Reprod 1999 5(10), 908]. FA1 has also been demonstrated in tumors[Jensen, Eur J Biochem 1994 225(1), 83-92; Tornehave, Histochem CellBiol 1996 106(6), 535; Harken Jensen, Tumour Biol 1999 20(5), Jensen,Mol Hum Reprod 1999 5(10), 908-913] including Small Cell Lung Cancer,pheochromocytomas and neuroblastomas.

Although FA1 expression has been observed in fetal muscle (Floridon etal., Differentiation 2000 66(1), 49-59) it was only noted to beexpressed in multi-nucleated myotubes of fetal skeletal muscles. Fullydifferentiated muscle cells were FA1 negative. Cardiac and smoothmuscles were also FA1 negative. There was no evidence of the antigen inmononuclear myogenic progenitor cells. Furthermore, to our knowledgethere are no published reports of FA1 expression in muscle stem- andprogenitor cultures derived from mammalian muscle.

Antibodies to FA1 have been used previously for cell sorting. Bauer,(Molecular And Cellular Biology, p. 5247-5255 Vol. 18, No. 9) usedanti-dlk1 polyclonal antiserum for dlk1 detection and flow cytometryanalysis of detached stromal cells and pre-B cells. Garćes(Differentiation 1999 64:103-114) used Pref-1 antibodies for flowcytometry analysis and cell sorting of preadipocytes and theirdifferentiated progeny.

BRIEF DESCRIPTION OF THE INVENTION

The present invention concerns the use of antibodies that recognize theFA1 antigen that is expressed as a membrane-associated protein inspecific populations of cells of mammalian muscle and in culturescontaining mammalian muscle stem- and progenitor cells. An example of anantibody used in the invention is the mouse monoclonal FA1 antibody(clone 142.2) or a mono-specific polyclonal anti-FA1 antibody. The FA1antibody binds to a population of mononuclear cells and immature musclecells present in mammalian muscle and to sub-populations of cells incultures containing and/or derived from muscle stem- and progenitorcells. The term mammalian includes any mammalian species, includingmouse, rat, domestic animals, and preferably human beings.

Accordingly, there is provided a method for obtaining a cell populationenriched in cells selected from the group consisting of musclemononuclear cells, satellite cells, muscle stem cells, and muscleprogenitor cells, said method comprising the steps of

-   -   a) providing a population of cells selected from the group        consisting of: a mixed population of mammalian muscle cells, and        cultures containing or derived from muscle stem- and/or        progenitor cells,    -   b) contacting said population with labeled antibodies which bind        specifically to FA1/dlk1,    -   c) selecting cells labelled with the FA1 antibody.

One advantage of using FA1/dlk for selection of cells is that dlk is acell surface protein and selection can be performed by simple methods ofantibody labelling, which result in the recovery of live cells which canbe used for further purposes such as culturing.

The invention also concerns a method for preparing a cell populationuseful for transplantation that is enriched in FA1-expressing musclemononuclear cells, satellite cells or muscle stem- or progenitor cells;which population may also be substantially free of other types of musclecells.

The invention also in one aspect concerns the differentiation and/ortransdifferentiation of such isolated myogenic stem/precursor cell intophenotypes distinct from the myogenic phenotype and used for replacementtreatment. Myogenic, osteogenic and adipogenic differentiation isdescribed in Asakura et al Differentiation 2001;68:245-253, which ishereby incorporated by reference. Another example would be(trans)differentiation along the hematopoietic lineages for replacementtherapy in leukemia where isolated hematopoietic stem cells from thepatient's own bone marrow may be complicated by contamination withcancer cells. Use of stem cells residing in the patient's muscle tissuewould diminish the risk of contamination with transformed blood cells.Methods for differentiation of muscle-derived stem cells intohematopoietic cells is described in Asakura et al J. Biol Chem;2002:123-134.

Differentiation of muscle stem cells or myogenic precursor cells mayinvolve the use of growth factors and also the use of low oxygen level,(below 12%, preferably from 1 to 5% oxygen) as described in U.S. Pat.No. 6,184,035, has been shown to enhance the differentiation intoskeletal muscle cells.

The invention also concerns therapeutic materials and methods fortransplanting cultures containing cells of the invention that can beused in the treatment of myodegenerative diseases such as Duchenne'sMuscular Dystrophy, and in the regeneration of muscle tissue followingtrauma, myocardial infarction or ischemia.

The present invention also provides cell populations enriched in FA1expressing muscle mononuclear cells, myogenic progenitor cells or musclestem- or progenitor cells, which are important vehicles for ex-vivo genetherapy. The cells may be obtained with the methods of the presentinvention. The cells may be encapsulated or implanted as naked cells.

These cell populations may also be used in drug screening, for thegeneration of cell-type specific antibodies and in gene discovery.

In a further aspect the invention relates to a method for measuring thecontent of FA1 expressing cells in a sample comprising the steps of:

-   -   a) contacting a population of cells selected from the group        consisting of: a mixed population of mammalian muscle cells,        populations of muscle stem- and/or progenitor cells, in vitro        differentiated muscle stem and/or progenitor cultures; with        labelled antibodies which bind specifically to FA1/dlk1,    -   b) optionally removing unbound antibodies and;    -   c) selecting cells labelled with the FA1 antibody,    -   d) quantifying the amount of selected cells resulting from        step (c) relative to the quantity of cells used in step (a).

This method may be used to quantify the regenerative capacity of a cellpopulation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1

Immunohistochemical staining for FA1/dlk1 in normal human fetal skeletalmuscle at gestational week a) 15 and b) 21 c) Staining of neonatal (0months) skeletal muscle. Arrows indicate mononuclear myogenic progenitorcells. Original magnifications, 400×

FIG. 2

Immunohistochemical staining for human FA1/dlk1 in an inflammatorymyopathy shown at two magnifications. Original magnifications: a) 200×and b)

FIG. 3

FA 1/dlk1 localization in cryosections of rat skeletal muscle duringregeneration of knife-cut lesioned muscle a)1, b) 3, c) 5, d) 7, e) 14and f) 32 days after the lesion was inflicted. Original magnifications:100×

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns the use of antibodies that can bind tothe FA1 antigen. More specifically it concerns the use of an antibody,referred to herein as “FA1 Ab” that facilitates the identification orisolation of specific populations of cells derived from mammalianmuscle. These isolated cell populations make possible improvedtechniques for transplantation, drug screening and gene discovery. Theisolated cells may also be employed to produce panels of monoclonalantibodies to specific populations of muscle-derived cells. Cellsexpressing FA1 are denoted FA1+ (FA1 positive) cells.

The isolated cell populations of the invention can also be employed inex vivo gene therapy. The isolated cell populations may be furthersorted based on the expression of other lineage specific markers.Examples include, but are not limited to; NCAM, VCAM1, Bcl-2, Pax7,Myosin, and M-cadherin. Co-selecting for one or more of these markers,improves the selection for myogenic precursor cells. Preferably theco-selecting also comprises a marker which is located on the cellsurface, such as NCAM, VCAM 1, and Myosin.

The ability to recognize myogenic progenitor cells with antibodiesallows not only for the identification and quantification of these cellsin tissue samples, but also for their separation and enrichment insuspension. This can be achieved by a number of cell-sorting techniquesby which cells are physically separated by reference to a propertyassociated with the cell-antibody complex, or a label attached to theantibody. This label may be a magnetic particle or a fluorescentmolecule. The antibodies may be cross-linked such that they formaggregates of multiple cells, which are separable by their density.Alternatively the antibodies may be attached to a stationary matrix, towhich the desired cells adhere.

It is evident that selection of FA1 expressing cells can also beobtained using labelled probes that bind selectively to the mRNAsequence of FA1. As however this mRNA is present within the cell suchlabelling is more complicated than selecting for the antigen located onthe surface of the cells and often would not be compatible withselecting viable cells because current methods for in situ hybridisationdo not allow for the recovery in large numbers of living cells. Afurther, less preferred, embodiment would be to transform the musclecells (preferably cultured cells) with a marker gene under the controlof the FA1 promoter. Such a marker protein would be co-expressed withFA1 and could be used for selection. Examples of suitable markerproteins include but are not limited to surface proteins, CD8, influenzavirus hemagglutinin, beta-galactosidase, green fluorescent protein,catachol 2,3-dioxygenase, and aeqourin. It suffices to use an expressionconstruct which gives rise to transient expression; stable integrationof the gene into the genome of the host cell is not required. Knowingthe sequence of the coding region of FA1 it is possible for the skilledperson to isolate the promoter sequence of FA1 and integrate it intosuch a construct.

In one embodiment, the present invention provides a method of selectinga population of cells derived from mammalian muscle; the methodcomprising: (a) providing a cell suspension from tissue derived frommuscle (b) contacting said cell suspension with an antibody that bindsthe FA1 antigen; and (c) separating and recovering from said cellsuspension the cells bound by said antibody, or (d) separating andrecovering from said cell suspension the cells not bound by saidantibody.

In a further embodiment the present invention provides a method ofselecting a population of FA1 expressing cells derived from cultures ofimmature muscle cells; the method comprising: (a) providing a cellsuspension prepared from a culture of immature muscle cells (b)contacting said cell suspension with an antibody that binds the FA1antigen; and (c) separating and recovering from said cell suspension thecells bound by said antibody, or (d) separating and recovering from saidcell suspension the cells not bound by said antibody.

In another embodiment the present invention provides a method ofselecting a population of FA1 expressing cells derived from cultures ofin-vitro differentiated immature muscle cells; the method comprising:(a) providing a cell suspension prepared from a culture of in-vitrodifferentiated immature muscle cells (b) contacting said cell suspensionwith an antibody that binds the FA1 antigen; and (c) separating andrecovering from said cell suspension the cells bound by said antibody,or (d) separating and recovering from said cell suspension the cells notbound by said antibody.

Yet another embodiment of the present invention provides populations ofmammalian muscle-derived cells enriched for FA1 expressing cells whichare mono-nuclear cells, satellite cells, muscle progenitor cells, musclestem cells, or immature muscle cells, which cultures may also besubstantially free of other types of mammalian cells, as well astherapeutic methods employing such a cell suspension. The various FA1+cell types may be present substantially alone or together with otherFA1+ cells. Such cultures may be transplanted into a donor by surgicalimplantation, injection, or by intra-venous infusion.

In a further embodiment, the invention provides cell populations usefulin methods of ex vivo gene therapy. Expression vectors may be introducedinto and expressed in these cells, or their genome may be modified byhomologous or non-homologous recombination by methods known in the art.In this way, diseases may be treated, which are related to the lack ofsecreted proteins including, but not limited to hormones, enzymes, andgrowth factors. Specific examples may include laminin, dystrophin andother factors known to affect muscle function. In one embodiment thecells may be used for treating mutations, i.e. introducing a functionalgene to replace the mutated in cells isolated using the methods of thepresent invention. Inducible expression of a gene of interest under thecontrol of an appropriate regulatory initiation region will allowproduction (and secretion) of the protein in a fashion similar to thatin the cell that normally produces the protein in nature. A furtherembodiment provides populations of mononuclear myogenic cells that havebeen immortalized by insertion of an immortalizing gene such as atelomerase or vmyc.

According to another embodiment of the invention, FA1+ cells can be usedin the production of monoclonal antibodies that recognize differentantigens on mono-nuclear cells, myogenic progenitor cells or immaturemuscle cells. The cells isolated from muscle with FA1 antibody can beused as an immunogen, as described below, to produce a panel ofmonoclonal antibodies against mononuclear cells, myogenic progenitorcells or immature muscle cells or against sub-populations of such cells.These monoclonal antibodies may in turn be used to identify furthermononuclear cells, myogenic progenitor cells or immature muscle cellsand to divide FA1+ cells into subpopulations.

Antibodies that label the populations of mono-nuclear cells, myogenicprogenitor cells or immature muscle cells and their differentiatedprogeny are extremely useful in drug screening, gene discovery and fortransplantation purposes because they allow the enrichment ofpopulations of such cells in a single step. Cells recovered with FA1antibody derived from different stages in their development could beused in studies on the mechanisms of action of cells, factors, and genesthat regulate cell proliferation and differentiation. Furthermore,myogenic progenitor cells from normal and pathological tissue may berecovered using FA1 antibodies and compared.

Production of Antibodies

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byone or more injections with the native protein, a synthetic variantthereof, or a derivative of the foregoing. An appropriate immunogenicpreparation can contain, for example, the naturally occurringimmunogenic protein, a chemically synthesized polypeptide resembling theimmunogenic protein, or a recombinantly expressed immunogenic protein.Furthermore, the protein may be conjugated to a second protein that isknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor.

The polyclonal antibodies directed against the immunogenic protein canbe isolated from the mammal (e.g. from the blood) and further purifiedby well known techniques, such as affinity chromatography, using proteinA or protein G, which provide primarily the IgG fraction of immuneserum. Subsequently, or alternatively, the specific antigen which is thetarget of the immunoglobulin sought, or an epitope thereof, may beimmobilized on a column to purify the immune specific antibody byimmunoaffinity chromatography.

Monoclonal anti-FA1 cell antibodies can be produced readily by oneskilled in the art. The general methodology for making monoclonalantibodies using hybridoma technology is now well known in the art. See,e.g., M. Schreier et al., Hybridoma Techniques (Cold Spring HarborLaboratory 1980); Hammerling et al., Monoclonal Antibodies and T-CellHybridomas (Elsevier Biomedical Press 1981); Kennett et al., MonoclonalAntibodies (Plenum Press 1980). Immortal, antibody-secreting cell linescan also be produced by techniques other than fusion, such as directtransformation of B-lymphocytes with oncogenic DNA or EBV. Severalantigen sources can be used, if desired, to challenge the normalB-lymphocyte population that is later converted to an immortal cellline.

The FA1 protein is expressed as a cell-surface antigen on many immaturecell populations. FA1 may also be purified from amniotic fluid as a32-38 kD glycoprotein. A purification method for mouse FA1 is given byBachmann et al., (J Reprod and Fert 1996; 107:279-285) and this can alsobe used for human FA1.

For example, the purified FA1 from amniotic fluid may be used as animmunogen to challenge the mammal (e.g., mouse, rat, hamster, etc.) usedas a source for normal B-lymphocytes. The antigen-stimulatedB-lymphocytes are then harvested and fused to an immortal cell line ortransformed into an immortal cell line by any appropriate technique. Apreferred hybridoma producing the monoclonal FA1 antibody is produced bychallenging a mouse with the FA1 antigen and fusing the recoveredB-lymphocytes with an immortal myeloma cell such as X63Ag8.6.5.3 orSP2/0-Ag14. Antibody-producing immortal cells can be screened forappropriate antibody production by selecting clones that are stronglyand specifically reactive with the muscle myogenic progenitor cellsusing sectioned muscle tissue and immunohistochemistry. Antibodiesproduced by clones, which show those properties can then be tested forreactivity towards other cell populations known to express FA1.

A mouse hybridoma producing monoclonal FA1 antibody (clone 142.2) isdescribed in a previous publication (Jensen et al., Eur J. Biochem. 1994Oct. 1; 225(1): 83-92.). Other Hybridomas producing FA1 antibodies are:F12, F15, F30, F59, F31, F32, F33, F38, F54, 142-1. These hybridoma areall available from the University of South Denmark, Institute of MedicalBiology, Winsløwsparken 21,1, DK-5000 Odense C, Denmark. These have beenobtained using the methods described in Jensen et al (op.cit.). Theimmunogen to be used for the generation of antibodies against FA1/dlk1may be 1) intact, native FA1 as purified from any human physiologicalfluid (milk, amniotic fluid, serum, seminal plasma, follicular fluid,urine); 2) FA1 or smaller products purified from primary cell culturesor cell lines (including genetically engineered cells) that generatesoluble dlk1 forms; 3) membrane fractions from cells that express allforms of dlk1; 4) synthetic peptides or fusion proteins encompassingparts of or the entire extracellular part of the multiple dlk1 forms or;5) chimeric proteins presenting any dlk1 form as a dimer, which includesfusion proteins and hybridoma cell lines in which the secretedimmunoglobulin molecule has been genetically modified so the Fab regionhas been replaced with dlk1 in any form.

The antibodies according to the subject invention may be eithermonoclonal, polyclonal, or a mixture of monoclonal and/or polyclonalantibodies. The antibody may comprise whole antibody or antigen-bindingfragments thereof, such as Fab₂, Fab and Fv fragments. Antigen bindingfragments can be prepared using conventional techniques known in theart, such as proteolytic digestion of antibody by papain or pepsin, orthrough standard genetic engineering techniques known in the art.Monoclonal antibodies exemplified herein can be engineered so as tochange the isotype of the antibody. For example, an IgG_(2A) isotype canbe engineered as an IgG₁, IgG_(2B), or other isotypes. Also contemplatedby the subject invention are antibodies that are reactive with the FA1antibody and which have been engineered to comprise human antibodyconstant regions. “Humanised” antibodies can be prepared using standardmethods known in the art. See, for example, U.S. Pat. No. 5,585,089(issued Dec. 17, 1996), the disclosure of which is hereby incorporatedby reference. Also contemplated are single chain antibodies and chimericantibodies.

Labelling of Antibodies

The antibodies of the subject invention can be labelled according tostandard methods known in the art. Preferably, the label is one capableof providing a fluorescent signal. Fluorescence is preferred due to thehigh signal/noise ratio. For example, antibodies can be labelled withdetectable labels such as fluorescein, rhodamine or with radioactiveisotopes, or with biotin. Biotin binds strongly and irreversible toavidin. Biotinylated antibodies may be visualized by incubation withconjugates consisting of horseradish perioxidase and biotin bound toavidin followed by detection of the enzymatic activity using achromogenic substrate. Alternatively, biotinylated antibodies may beincubated with a streptavidin-flurochrome.

Isolation of FA1 Expressing Cells

As indicated above, one application for antibodies to FA1 is theisolation of an enriched source of myogenic progenitor cells fortransplantation into patients with Duchene's Muscular Dystrophy, orfollowing trauma or myocardial infarction

The present invention contemplates the use of methods employing a FA1antibody to separate muscle myogenic progenitor or progenitor cells fromother muscle cells. The cells used for isolation include skeletal(striated) muscle, cardiac muscle and smooth muscle. These may originatefrom fetal tissue or adult tissue. The cells may also originate fromcell cultures of raised from either of the above-identified cell types,such as cell cultures raised from embryonal stem cells.

Generally, a cell suspension prepared from mammalian muscle tissue bymechanical or enzymatic trituration is brought into contact with a FA1antibody. Cells that have been bound by FA1 antibody are then separatedfrom unbound cells by any means known to those skilled in the art. Themuscle tissue may be taken from any muscular region or organ and may beselected by dissection. For instance it may be taken from skeletal(striated) muscle, cardiac muscle, or smooth muscle.

Various methods of separating antibody-bound cells from unbound cellsare known. For example, the antibody bound to the cell (or ananti-isotype antibody) can be labeled and then the cells separated by amechanical cell sorter that detects the presence of the label.Fluorescence-activated cell sorters are well known in the art. In oneembodiment, the anti-FA1 antibody is attached to a solid support.Various solid supports are known to those of skill in the art,including, but not limited to, agarose beads, polystyrene beads, hollowfiber membranes, polymers, and plastic petri dishes. Cells that arebound by the antibody can be removed from the cell suspension by simplyphysically separating the solid support from the cell suspension.Preferred protocols, however, will be described.

Most of the isolation methods described and used in the art comprise astep of removing unbound antibodies prior to selecting cells. Howeverdepending on the sensitivity of the detection method, the strength ofthe signal originating from the label, and the number of FA1 moleculeson the surface of the cells this step may not be possible. Important isthat it is possible to distinguish FA1 expressing cells from backgroundand from non-FA1 expressing cells.

Super paramagnetic nanoparticles may also be used for cell separations.The microparticles are coated with a monoclonal antibody for acell-surface antigen. The antibody-tagged, super paramagneticmicroparticles are then incubated with a solution containing the cellsof interest. The microparticles bind to the surfaces of the desiredcells, and these cells can then be collected in a magnetic field.

Selective cytophoresis can be used to produce a cell suspension frommammalian muscle containing myogenic progenitor cells. The cellsuspension is allowed to physically contact, for example, a solidphase-linked monoclonal antibody that recognizes an antigen on thedesired cells. The solid-phase linking can comprise, for instance,adsorbing the antibodies to a plastic, nitrocellulose, or other surface.The antibodies can also be adsorbed on to the walls of the large pores(sufficiently large to permit flow-through of cells) of a hollow fibermembrane. Alternatively, the antibodies can be covalently linked to asurface or bead, such as Pharmacia Sepharose 6 MB macrobeads. The exactconditions and duration of incubation for the solid phase-linkedantibodies with the muscle cell suspension will depend upon severalfactors specific to the system employed. The selection of appropriateconditions, however, is well within the skill of the art.

The unbound cells are then eluted or washed away with physiologic bufferafter allowing sufficient time for the stem cells to be bound. Theunbound cells can be recovered and used for other purposes or discardedafter appropriate testing has been done to ensure that the desiredseparation had been achieved. The bound cells are then separated fromthe solid phase by any appropriate method, depending mainly upon thenature of the solid phase and the antibody. For example, bound cells canbe eluted from a plastic petri dish by vigorous agitation.Alternatively, bound cells can be eluted by enzymatically “nicking” ordigesting an enzyme-sensitive “spacer” sequence between the solid phaseand the antibody. Spacers bound to agarose beads are commerciallyavailable from, for example, Pharmacia.

The eluted, enriched fraction of cells may then be washed with a bufferby centrifugation and either said enriched fraction or the unboundfraction may be cryopreserved in a viable state for later use accordingto conventional technology or introduced into the transplant recipient.

The term ‘enriched’ is used to describe a population of cells in whichthe proportion of one particular cell type or the proportion of a numberof particular cell types is increased when compared with the untreatedpopulation. According to one embodiment of the invention a populationenriched in FA1+ cells comprises at least 5% FA1+ cells, more preferablyat least 10%, more preferably at least 20%, more preferably at least25%, more preferably at least 30%, such as at least 40%, for example atleast 50%, such as at least 60%, for example at least 75%, such as atleast 80%, more preferably at least 90%, more preferably at least 95%,more preferably at least 98%, such as substantially 100% FA 1+ cells.

The above cell populations containing FA1 enriched cells can be used intherapeutic methods such as cell transplantation, as well as othermethods that are readily apparent to those skilled in the art. Otheruses envisaged for these cells are for drug screening, antibodyproduction and gene discovery. The compositions of the invention mayalso be used to generate antibodies to the membrane bound portion ofdlk1, which remains following proteolytic cleavage of dlk1 to give thesoluble form. They may also be used in methods to identify the proteaseresponsible for cleavage of dlk1. For example dlk1 could be expressed ina eukaryotic cell (e.g. yeast) that does not normally process it. Theeukaryotic cell could then be contacted with fractionated cell extractsfrom FA1 producing cells, and the fraction which cleaves dlk1could beidentified and treated to isolate the said protease. The protease, whichcleaves dlk1, could be a key element in the differentiation of primitivecell types. It is also envisaged that fractionated extracts containingthe protease, obtained from enriched populations of FA1 producing cellsof the invention, could be used to regulate the differentiation of stem-and progenitor cells.

It is understood that the initial medium for isolating stemcells/progenitor cells, the medium for proliferation of these cells, andthe medium for differentiation of these cells can be the same ordifferent. All can be used in conjunction with low or physiologic oxygenlevel culturing. The medium can be supplemented with a variety of growthfactors, cytokines, serum, etc. Examples of suitable growth factors arebasic fibroblast growth factor (bFGF), vascular endothelial growthfactor (VEGF), epidermal growth factor (EGF), transforming growthfactors (TGFα and TGFβ), platelet derived growth factors (PDGF's),hepatocyte growth factor (HGF), insulin-like growth factor (IGF),insulin, erythropoietin (EPO), and colony stimulating factor (CSF).Examples of suitable hormone medium additives are estrogen, progesteroneor glucocorticoids such as dexamethasone. Examples of cytokine mediumadditives are interferons, interleukins, or tumor necrosis factor-α(TNFα).

In general, populations of FA1 positive myogenic progenitor cells may beused to establish primary cell cultures which can be expanded and usedfor transplantation, drug screening or any of the other purposesmentioned above. In one embodiment, populations of FA1 positive myogenicprogenitor cells may be treated with the soluble FA1 antigen in order tomaintain them in an undifferentiated state. This can be done with nativehuman FA1 used in quantities from 1 to 10 μg/mL, such as approximately 5μg/mL. Further details on the use of soluble FA1 to maintain anundifferentiated state are available in Hansen et al, Mol. Endocrinol1998; 12:1140-49.

In another embodiment, the FA1 antibody can be used to isolate FA1enriched cells, which can be used in various protocols of genetictherapy.

EXAMPLES

The following examples are provided to illustrate specific embodimentsof the present invention. The examples are included for illustrativepurposes only, and are not intended to limit the scope of the presentinvention.

Example 1

FA1/dlk1 in Human Fetal Muscle.

Human tissues: Normal fetal (n=6, gestational week 12-23) and neonatal(n=2, age=0 and 2 months) striated muscle tissue samples were obtainedfrom the files at the Department of Pathology, Odense UniversityHospital. Formalin fixed and paraffin embedded human muscle specimenswere cut in 5 μm sections, mounted on glass slides; air-dried andsubsequently deparaffinized and re-hydrated. Endogenous peroxidaseactivity was blocked with H₂O₂ /methanol. Antigen-retrieval wasperformed by incubation with 0.05% (w/v) protease (Sigma, type XIV) inTBS at 37° for 15 minutes. Sections were incubated with a primaryantibody (monospecific rabbit anti-human FA1) or a control antibody(primary antibody liquid-phase absorbed with affinity purified human FA1as described in Jensen et al., 1993) diluted 1:100 (anti-human FA1) andsubsequently reacted with a biotinylated secondary antibody (goatanti-rabbit IgG (DAKO E432, diluted 1:200)). The sections were thenincubated with HRP-conjugated streptavidin (DAKO P397) diluted 1:300 anddeveloped using 3-amino-9-ethylcarbazol as chromogen. Counter-stainingwas performed with haematoxylin.

The sections demonstrated FA1 immuno-reactivity in the muscle fibers(FIGS. 1 a and 1 b). The intensity of the reaction decreased withincreasing age. An accentuated perinuclear reaction was common. Inaddition, the mononuclear cells of spindle type situated both adjacentto the muscle fibers and more solitarily situated between the fibersexpressed FA1. Their staining intensity was higher than that of thefibers and remained unaltered form 12 to 23 weeks of gestation, though areduction in number seemed to take place.

In the neonatal muscle the muscle fibers were unstained but scattered,densely stained, mononuclear cells adjacent to the fibers were found; incontrast to earlier gestational ages where all such cells were FA1immuno-reactive. Their position adjacent to the fibers suggests thatthey belong to the myogenic progenitor cell population (FIG. 1 c).

Example 2

FA1/dlk1 in Adult Human Striated Muscle.

Muscle tissue samples from 6 individuals with an inflammatory myopathywere obtained from the files at the Department of Pathology, OdenseUniversity Hospital. Normal adult skeletal muscle was obtained frombiopsies with approval from the regional science ethical committee forVejle and Funen counties.

Tissues were formalin-fixed and paraffin embedded and stained as inexample 1. In normal adult human skeletal muscle no FA1 immuno-reactionwas observed. By contrast, in a series of 6 inflammatory myopathies allcharacterized by containing inflammatory infiltrates and necrotic andregenerating muscle fibers, mononuclear FA1 positive cells were present.They were located in close relation to apparently intact muscle fibersbut not found at sites of necrosis (FIG. 2).

Example 3

FA1/dlk1 Expression in a Rat Muscle Lesion Model.

Normal adult rat skeletal muscle was obtained from carbon monoxide (CO)intoxicated and decapitated Sprague-Dawley rats (M&B, Denmark). Animalexperiment: Adult male rats (n=23) were deeply anaesthetized withpentobarbital and a knife cut lesion inflicted in the (thigh muscle).Animals were sacrificed by CO2-intoxication either 2 hours, 1, 3, 5, 7,14, 32 or 56 days after the injury was inflicted and the lesioned musclewas removed. All rat specimens were quick-frozen in isopentane andstored at −70° C. until further analyzed. Cryosections (5 μm) of ratmuscle specimens were air-dried overnight and fixed in acetone for 10min at room temperature. Sections were incubated with a primary antibody(monospecific rabbit anti-rat FA1) or a control antibody (primaryantibody liquid-phase absorbed with affinity purified rat FA1 asdescribed in Jensen et al., 1993) diluted 1:2000 and subsequentlyreacted with a biotinylated secondary antibody (goat anti-rabbit IgG(DAKO E432, diluted 1:200)). The sections were then incubated withHRP-conjugated streptavidin (DAKO P397) diluted 1:300 and developedusing 3-amino-9-ethylcarbazol as chromogen. Counter-staining wasperformed with haematoxylin.

Normal adult rat muscle contained single scattered FA1-positive cells inapposition to muscle fibers in some areas, but large areas wereFA1-negative (not shown). However, we observed that knife-cut lesionsinduced an upregulation in the expression of FA1/dlk1 In order toinvestigate the time sequence of the FA1-induction in regeneratingmuscle a series of rat muscles with lesions varying from 2 h to 56 dayswere studied; Two hours after introduction of a cut lesion no FA1immuno-reactivity had developed in relation to the lesion. However, atday 1 FA1-positive mononuclear cells situated along the damaged fibersappeared (FIG. 3 a) and at day 3 an intense staining in these cells haddeveloped in a zone around the lesion. A few cells of this type werefound at the edge of the lesion but not within its center (FIG. 3 b). Atday 5 (FIG. 3 c) the zone around the lesion containing FA1immuno-reactive cells was narrower but more densely packed. Scatteredless densely stained cells could be found at the rim of the lesion, butat the cut ends where fusion could be identified as newly formed musclefibers with irregular contours and disorderly arranged nuclei, noFA1-positive mononuclear cells were found. The newly formed segments ofmuscle fibers were FA1-negative at this and any other age of lesion.Except for a further reduction of the peri-lesional zone containing FA1immuno-reactive cells no changes were seen between day 5 and 7 (FIG. 3d).

At day 14 a reduction of the number of stained cells in theperi-lesional zone and also a reduction in their staining intensitycould be observed. No FA1-positive cells were found in the fusion zoneat this stage of regeneration (FIG. 3 e).

At day 32 only few FA1 immuno-reactive cells remained detectable (FIG. 3f) and at day 56 all had disappeared (not shown). No structures exceptthe mononuclear cells in apposition to the muscle fibers expressed FA1.

Example 4

FA1/dlk1 Expression in Cultures.

Satellite cell cultures were established according to Gaster et al.(APMIS 2001:109(11):726-734). In brief, human muscle tissue was minced,washed, and enzymatically dissociated for 60 min. with 0.05%Trypsin-EDTA. Harvested cells were preplated in DMEM with 10% FCS andantibiotics on uncoated tissue culture plates for 30 min. Non-adherentcell were next transferred to culture dishes coated with 1% ECM-gel.After 24 hours, the medium was changed to DMEM supplemented with 2%Ultroser-G and 2% FCS. Before confluence was reached, the adherent cellswere trypsinized, preplated as described for 30 min. and thenon-adherent cells transferred to new coated culture plates. Thisprocedure was repeated until the primary culture underwent four passagesto ensure that all fibroblasts were removed form the cell population.

For studies on differentiating satellite cells, the established primaryculture, devoid of fibroblasts, was plated onto non-coated tissueculture plates. At 75% confluence, the medium was replaced with basalmedium (DMEM, antibiotics, and 25 pM insulin) containing 2% FCS (Gasteret al., APMIS 2001;109(11):735-744).

Primary and differentiated cultures were stained for FA1/dlk1 using theantibodies described. In addition, initially harvested cells and primaryand differentiated cultures were analyzed by FACS using the sameantibodies.

1. A method for obtaining a cell population enriched in cells selectedfrom the group consisting of muscle mononuclear cells, satellite cells,muscle stem cells, and muscle progenitor cells, said method comprisingthe steps of a) contacting a population of cells selected from the groupconsisting of: a mixed population of mammalian muscle cells, andcultures containing or derived from muscle stem- and/or progenitorcells, with labeled antibodies which bind specifically to FA1/dlk1, b)selecting cells labelled with the FA1 antibody.
 2. The method of claim1, further comprising the step of removing unbound antibodies prior tothe step of selecting cells.
 3. The method of claim 1 wherein the cellpopulation is enriched in FA1+ muscle mono-nuclear cells.
 4. The methodof claim 1 wherein the cell population is enriched in FA1+ musclesatellite cells.
 5. The method of claim 1 wherein the cell population isenriched in FA1+ muscle progenitor cells.
 6. The method of claim 1,wherein the cell population is enriched in FA1+ muscle stem cells. 7.The method of claim 1 wherein the cell population is enriched in one ormore FA1+ cell types, the cell types being selected from: musclemononuclear cells; muscle satellite cells; muscle progenitor cells, andmuscle stem cells.
 8. The method according to claim 1, wherein the cellpopulation obtained has been further selected for expression or lack ofexpression of at least one further marker.
 9. The method according toclaim 8, wherein the marker is selected from the group consisting ofNCAM, VCAM1, M-cadherin, Bcl-2, Pax7 and Myosin.
 10. The methodaccording to claim 8, wherein the marker is NCAM.
 11. The methodaccording to claim 8, wherein the marker is Pax7.
 12. The methodaccording to claim 1, wherein the cells are human cells.
 13. The methodaccording to claim 1, wherein the cells are fetal cells.
 14. The methodaccording to claim 1, wherein the cells are taken from skeletal(striated) muscle, cardiac muscle, or smooth muscle.
 15. The methodaccording to claim 1, wherein the antibodies are fluorescently labelled.16. The method according to claim 1, wherein the antibodies aremagnetically labelled.
 17. The method according to claim 1, wherein theantibodies are cross-linked.
 18. The method according to claim 1,wherein the antibodies are biotin labelled.
 19. A method fordifferentiation of myogenic stem/precursor cells comprising isolatingFA1+ cells according to the method of claim 1, and furtherdifferentiating these cells into phenotypes distinct from the myogenicphenotype.
 20. A method for transdifferentiation of myogenicstem/precursor cells comprising isolating FA1+ cells according to themethod of claim 1, and further transdifferentiating these cells intophenotypes distinct from the myogenic phenotype.
 21. The method of claim20, wherein the cells are transdifferentiated along the hematopoieticlineage.
 22. A composition of cells derived from mammalian muscle, whichis enriched for FA1⁺ mono-nuclear cells/myogenic progenitor cells ormuscle stem- or progenitor cells.
 23. A composition of cells obtained bya process comprising the steps of: a) contacting a population of cellsselected from the group consisting of a mixed population of mammalianmuscle cells, and cultures containing or derived from muscle stem-and/or progenitor cells, with labeled antibodies which bind specificallyto FA1/dlk1, b) selecting cells labelled with the FA1 antibody.
 24. Agenetically modified cell generated from the composition of claim 22.25. The genetically modified cell of claim 24, which has been modifiedto express at least one hormone, enzyme, and/or growth factor. 26-35.(canceled)
 36. A method for measuring the content of FA1 expressingcells in a sample comprising the steps of: a) contacting a population ofcells selected from the group consisting of: a mixed population ofmammalian muscle cells, populations of muscle stem- and/or progenitorcells, in vitro differentiated muscle stem and/or progenitor cultures;with labelled antibodies which bind specifically to FA1/dlk1; b)optionally removing unbound antibodies; c) selecting cells labelled withthe FA1 antibody; and d) quantifying the amount of selected cellsresulting from step (c) relative to the quantity of cells used in step(a).
 37. The method according to claim 36, further comprising the stepof selecting the cells for expression or lack of expression of at leastone further marker.
 38. A method of identifying mono-nuclear myogeniccells comprising contacting the cells with an antibody to FA1/dlk1 andimaging the antibody.